Among the most significant discoveries of the last decade was that of RNA-based mechanisms of post-transcriptionally regulating gene expression (Hamilton, et al., Nature 431:371-378 (2004); Fire, et al., 391:806-811 (1998); Zamor, et al., Cell 101:25-33 (2000). It has been found that, when double stranded RNA enters cells, e.g., due to viral infection, it is recognized and cleaved by specific “dicer enzymes” into fragments (siRNAs) 21-25 nucleotides in length with 3′ dinucleotide overhangs. These fragments bind to a protein complex (RISC) that causes the RNA to unwind and one of the strands, the passenger strand, to be degraded. The remaining sequence targets the complex to an mRNA having a complementary sequence which is then cleaved. As a result, the expression of protein encoded by that mRNA is prevented.
Primitive cells and organisms may have used this system to protect themselves from virus infection long before the development of adaptive immune systems. Higher organisms appear to have adapted the system to modulating gene expression. In humans for example, endogenous genomic sequences transcribe RNA capable of folding back on itself to form double stranded regions. These are cleaved by enzymes to form “miRNA” fragments that then act in essentially the same manner as siRNAs. However, unlike siRNAs, the miRNAs often contain mismatches that do not allow the cleavage of mRNA targets. Rather, partially complementary target mRNAs are deadenylated, decapped, and degraded by the 5′-3′ exonucleolytic pathway. Alternatively, these mRNAs can be subject to translational silencing by a mechanism that is poorly characterized (for overview see, Ambros, Nature 431:350-355 (2004); Mattick, EMBO Reports 2:986-991 (2001); Bentwick, et al., Nature Genetics 37:766-770 (2005)).
The process of catalytically shutting down the translation of specific mRNAs by introducing double stranded RNA into cells can be used to target essentially any chosen target transcript. Thus, RNA interference is a technique of great interest clinically (where genes associated with diseases may be targeted) and to researchers attempting to identify the function of genes, e.g., resulting from the human genome project (see generally U.S. Pat. Nos. 7,232,806; 7,078,196 and 7,056,704).
Posttranscriptional regulation of gene expression plays an especially important role in the survival of mammalian cells exposed to adverse environmental conditions (Ron, et al., Nature reviews 8:519-529 (2007)). Control is accomplished by the downregulation of protein synthesis due to the phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (Hershey, Annu. Rev. Biochem. 60:717-755 (1991)). In addition, recent evidence has indicated the existence of a separate, phospho-eIF2α independent, translation control pathway (McEwen, et al., J. Biol. Chem. 280:16925-16933 (2005); Tenson, et al., Mol. Microbiol. 59:1664-1677 (2006); Rocha, et al., Food Add. Contam. 22:369-378 (2005); Iordanov, et al., J. Biol. Chem. 273:15794-15803 (1998); Shifrin, et al., J. Biol. Chem. 274:13985-13992 (1999)).
In Tetrahymena thermophila, nutrient stress induces cleavage of the tRNA anticodon loop to produce RNA fragments derived from the 5′ and 3′ ends of most, if not all, tRNAs (Lee, et al., J. Biol. Chem. 280:42744-42749 (2005)). The 3′ fragments of these tiRNAs lack the terminal CCA residues required for aminoacylation, suggesting that anticodon cleavage occurs following 3′ end processing, but prior to CCA addition. In mammalian cells, analogous RNAs comprise a small subset of piwi-associated piRNAs suggesting that tRNA anticodon cleavage may be a widespread phenomenon that can lead to the assembly of specific RNP complexes (Brennecke, et al., Cell 128:1089-1103 (2007); Grivna, et al., Proc. Nat'l Acad. Sci. USA 103:13415-13420 (2006); Lau, et al., Science 313:363-367 (2006)).